Previous work from this laboratory has revealed the presence of an exon that can be spliced into the heavy chain of nonmuscle myosin II-B (NMHC II-B) in loop I near the ATP binding domain. Although NMHC II-B has a wide cellular distribution throughout the body, the inserted isoform is only expressed in neuronal cells. NMHC II-C also contains an alternative exon composed of 24 nucleotides encoding 8 amino acids (C1). This exon, similar to that present in NMHC II-B, is spliced into loop I, but unlike the neuronal-specific II-B isoform, is present in a wide variety of mouse tissues. These include liver, kidney and testis, where RT-PCR analysis reveals that the mRNA encoding the inserted form is greater than 90% compared to the noninserted isoform, as well as brain and lung, where it is about 50%. Adult mouse skeletal and cardiac muscle tissues are devoid of the inserted NMHC-IIC isoform, but human skeletal muscle NMHC II-C mRNA is more than 50% inserted isoform. The expression of the inserted isoform changes during mouse development, being 50% at embryonic stage 14 and rising to approximately 90% after birth in the liver and kidney. The mouse cerebrum contains less of the inserted isoform at E14 (approximately 20%), but increases to 50% one week after birth. On the other hand, mouse skeletal muscle expresses over 90% of the inserted isoform at E14, which decreases to 0% after birth. A number of cell lines have also been examined, including mouse C2C12 (after differentiation, 100% noninserted isoform), rat PC12 (100% inserted) and Human HepG2 cells (100% inserted). We have cloned both isoforms of NMHC II-C fused with GFP protein to study their subcellular localization in Hela cells. They are found to localize at the periplasm of the cell and at the cytokinetic ring during cell division of Hela cells. Time-lapse videomicroscpy of Hela cells during cytokinesis clearly indicates that II-C is present until the complete generation of two daughter cells. When this inserted form of II-C was decreased by RNAi transfection in a human lung cancer cell line (A-549), the cells showed growth and migration defects, but no multinucleation as seen following down-regulation of NMHC II-B in COS-7 cells. Presently, we are generating C1 insert knockout mice to investigate the function of this 8 amino acid during mouse development. We have already generated the necessary chimeras and are presently checking for germline transmission.